Sugar mixtures and methods for production and use thereof

ABSTRACT

A sugar mixture comprising: monosaccharides; oligosaccharides in a ratio ≧0.06 to total saccharides; disaccharides in a ratio to total saccharides ≧0.05; pentose in a ratio to total saccharides ≧0.05; at least one alpha-bonded di-glucose; and at least one beta-bonded di-glucose. Also disclosed are methods to make and/or use such mixtures.

RELATED APPLICATIONS

In accord with the provisions of 35 U.S.C. §119(e) and §363, thisapplication claims the benefit of:

U.S. 61/358,894 filed 26 Jun. 2010 by Aharon EYAL and entitled“Fermentation Feedstock Precursor and Methods for the ProductionThereof”; and

U.S. 61/491,243 filed 30 May 2011 by Robert JANSEN et al. and entitled“Lignin Compositions, Systems and Methods for Processing Lignin and/orHCl”; and

U.S. 61/500,169 filed 23 Jun. 2011 by Aharon EYAL et al. and entitled“Sugar Mixtures and Methods for Production and Use thereof”;

In accord with the provisions of 35 U.S.C. §119(a) and/or §365(b), thisapplication claims priority from:

prior Israeli application IL206896 filed on 8 Jul. 2010 by Aharon EYALand entitled “Fermentation Feedstock Precursor and Methods for theProduction Thereof”: and

prior Israeli application IL207313 filed on 29 Jul. 2010 by Aharon EYALet al. and entitled “Methods for the Production of a FermentationFeedstock”; and

prior PCT application IL2011/000130 filed on 6 Feb. 2011 by Aharon EYALet al. and entitled “Methods for the Separation of HCl from aCarbohydrate and Compositions Produced thereby” which corresponds to IL210998 filed 1 Feb. 2011.

Each of these priority documents is fully incorporated by reference.

This application is also related to the following co-pendingapplications which are each fully incorporated herein by reference:

U.S. 61/473,134 filed 7 Apr. 2011 by Aharon EYAL and entitled“Lignocellulose Conversion Processes and Products”; and

U.S. 61/483,663 filed 7 May 2011 by Aharon EYAL and entitled“Lignocellulose Conversion Processes and Products”

U.S. 61/483,777 filed 9 May 2011 by Robert JANSEN et al. and entitled“Hydrolysis Systems and Methods”; and

U.S. 61/487,319 filed 18 May 2011 by Robert JANSEN et al. and entitled“Hydrolysis Systems and Methods”; and

prior Israeli application IL 211093 filed on 6 Feb. 2011 by Aharon EYALand entitled “A Method for Processing a Lignocellulosic Material and forthe Production of a Carbohydrate Composition”

FIELD OF THE INVENTION

This invention relates to sugars and production and use thereof.

BACKGROUND OF THE INVENTION

The carbohydrate-conversion industry is large and rapidly increasing insize. Currently, about 100 million tons of carbohydrates are fermentedannually, primarily to provide fuel-grade ethanol. This number ispredicted to triple in the next decade.

Millions of tons of carbohydrates are also fermented every year toprovide food and feed products, such as citric acid and lysine. Alsolarge and increasing is fermentation to produce other products, such asmonomers for the polymer industry, e.g. lactic acid for the productionof polylactide.

Fermentation media typically include, in addition to carbohydratesand/or another carbon source, other nutrients and factors such asnitrogen sources, minerals, vitamins, and growth factors. In some cases,fermentation media comprise well identified chemicals. In other cases,various preparations (e.g. yeast extract or tryptone broth) areincorporated without fully understanding the effect of each component inthe preparation. Some of those preparations result from natural sources,such as extracts. Some of those preparations are of relatively highcost.

With the advent of molecular biology techniques, a new generation ofindustrial fermentation, also known as conversion, based upongenetically modified microorganisms has emerged. In some cases thesemicroorganisms rely upon inducible promoters for induction of a specificgene. Some of the inducible promoters respond to specific sugars.

Although conversion of lignocellulosic material to carbohydrates viaenzyme-catalyzed and/or acid-catalyzed hydrolysis of polysaccharides andpyrolysis of lignocellulosic material have been previously described,industrial scale application of the proposed technologies has presentedtechnical problems which remain to be overcome. Hydrolysis ofhemicellulose is relatively easy, but hydrolysis of cellulose (typicallymore than 50% of total polysaccharides) is more difficult due to itspartial crystalline structure.

This application refers to various solvents defined in terms of Hoy'scohesion parameter Delta-P and/or Delta-H. By way of review:

Delta-P is the polarity related component of Hoy's cohesion parameterand delta-His the hydrogen bonding related component of Hoy's cohesionparameter.

The cohesion parameter, as referred to above or, solubility parameter,was defined by Hildebrand as the square root of the cohesive energydensity:

$\delta = \sqrt{\frac{\Delta \; E_{vap}}{V}}$

where ΔE_(vap) and V are the energy or heat of vaporization and molarvolume of the liquid, respectively. Hansen extended the originalHildebrand parameter to a three-dimensional cohesion parameter.According to this concept, the total solubility parameter, delta, isseparated into three different components, or, partial solubilityparameters relating to the specific intermolecular interactions:

δ²=δ_(d) ²+δ_(p) ²+δ_(h) ²

in which delta-D, delta-P and delta-H are the dispersion, polarity, andHydrogen bonding components, respectively. Hoy proposed a system toestimate total and partial solubility parameters. The unit used forthose parameters is MPa^(1/2). A detailed explanation of that parameterand its components can be found in “CRC Handbook of SolubilityParameters and Other Cohesion Parameters”, second edition, pages122-138. That and other references provide tables with the parametersfor many compounds. In addition, methods for calculating thoseparameters are provided.

SUMMARY OF THE INVENTION

One aspect of some embodiments of the invention relates to heterogeneoussugar mixtures. The term “sugar” as used in this specification and theaccompanying claims refers to monosaccharides and oligosaccharides(disaccharides or greater) soluble in water at 25 degrees centigrade.Throughout this application, the term disaccharide refers to sugardimers and “higher oligosaccharides” or “higher saccharide” refers tooligomers comprising three or more sugar units. According to variousexemplary embodiments of the invention dimers may be homo-dimers and/orhetero-dimers. Alternatively or additionally, higher oligosaccharidesmay include same and/or different sugar units.

In many exemplary embodiments of the invention, the sugar mixturesresult from acid hydrolysis of lignocellulosic or “woody” substrates. Insome exemplary embodiments of the invention, acid hydrolysis isconducted with HCl, optionally at a concentration of 37% W/WHCl/[HCl+water], optionally 39, 41, 43 or even 45% HCl on the samebasis. In some exemplary embodiments of the invention, the hydrolysis isconducted at temperatures below 50 degrees centigrade, optionally below40 degrees, optionally below 30 degrees, optionally at 25 degrees orless. In some exemplary embodiments of the invention, at least a portionof the hydrolysis is conducted at 20 degrees or less, optionally 15degrees or less.

According to various exemplary embodiments of the invention the sugarmixture is provided as a syrup and/or liquid, optionally includingresidual acid from hydrolysis. Total sugar concentrations in such asyrup/liquid can be 15, 20, 25, 30, 35, 40, 45 or 50% by weight orintermediate or higher percentages. Optionally, the mixture is providedas dry crystals.

In some exemplary embodiments of the invention, a percentage ofoligosaccharides to total saccharides in the mixture is greater than 4,optionally 6, optionally 8, optionally 10, optionally 15% or interveningor greater percentages.

In some exemplary embodiments of the invention, a percentage ofdisaccharides to total saccharides in the mixture is greater than 3,optionally 5, optionally 7, optionally 10% or intervening or greaterpercentages.

“Disaccharide” indicates two sugars connected by an alpha bond or by abeta bonds or bonded via various hydroxyls on the molecule, andcombinations thereof.

In some exemplary embodiments of the invention, a percentage of pentosesto total saccharides in the mixture is greater than 3, optionally 5,optionally 7, optionally 10% or intervening or greater percentages.Optionally, at least a portion of the pentose is present as part of adisaccharide or longer oligosaccharide.

According to various exemplary embodiments of the invention the mixtureincludes at least one alpha-bonded di-glucose and/or at least onebeta-bonded di-glucose. Optionally, at least a portion of thedi-glucoses are present as parts of higher oligosaccharides.

In some exemplary embodiments of the invention, residual acid (e.g. HCl)may be present in the mixture. Various exemplary embodiments of theinvention are concerned with ways to remove this residual acid.Optionally, such removal contributes to added value for use indownstream processes (e.g. fermentation). In some exemplary embodimentsof the invention, removal of residual acid involves extraction with anextractant containing an alcohol. Optionally, two or more extractionsare conducted. In some exemplary embodiments of the invention, at leastone of the extractions employs a mixture of two solvent types.

Another aspect of some embodiments of the invention relates tohydrolyzing a lignocellulosic substrate in HCl to form a hydrolyzatecomprising total saccharides to (total saccharides+water) of at least20%, optionally 25%, optionally 30% by weight and de-acidifying thehydrolyzate while increasing the sugar concentration. Optionally, thesugar concentration is increased to 35, 40, 45 or 50% or greater orintermediate percentages. In some exemplary embodiments of theinvention, the disaccharides in the de-acidified hydrolyzate are atleast 5%, optionally 10%, optionally 20%, optionally 30% of the totalsaccharides or intermediate or greater percentages. Optionally, aportion of the saccharides in the de-acidified hydrolyzate can beenzymatically digested.

Another aspect of some embodiments of the invention relates tofermenting such a sugar mixture in a fermentor to produce a desiredfermentation product or “conversion product”. Optionally, thefermentation product can include one or more of alcohols, carboxylicacids, amino acids, monomers for production of industrially importantpolymers and proteins. In some exemplary embodiments of the invention, afermentation product is produced and then converted into the monomer(e.g. 3-hydroxy-propionic acid to be converted into acrylic acid, whichis then polymerized). In other exemplary embodiments of the invention,the monomer is produced directly (e.g. lactic acid as a source ofpolylactide).

Optionally, the proteins are heterologous proteins produced bygenetically modified microorganisms. Such heterologous proteins include,but are not limited to, hormones, enzymes (e.g. cellulases), growthfactors, cytokines and antibodies. Optionally, the antibodies are fusionproteins including a non-immunoglobulin domain.

An additional aspect of some embodiments of the invention relates toenzymatic hydrolysis of a portion of the sugars in the mixture. Forpurposes of this specification and the accompanying claims, the term“enzyme” indicates a single enzyme or a mixture including two or moreenzymes. Optionally, an enzyme is provided as a crude preparation (e.g.cell extract) characterized by a type and/or level of activity, asopposed to a precise molecular definition. According to variousexemplary embodiments of the invention enzymes capable of hydrolyzingalpha and/or beta bonds are used. Optionally, specificity for a desiredbond type can be achieved by appropriate enzyme selection and/orselection of suitable reaction conditions. In some exemplary embodimentsof the invention at least 10% of disaccharides in the mixture areconverted to monosaccharides by this enzymatic treatment. Alternativelyor additionally, at least 10% of oligosaccharides in the mixture areenzymatically hydrolyzed to release additional monosaccharides. In someexemplary embodiments of the invention, enzymes are immobilized.Optionally, immobilization can be on beads and/or a membrane. In someexemplary embodiments of the invention, immobilization contributes to anincrease in yield of an enzymatic hydrolysis product per unit of enzyme.

For purposes of this specification and the accompanying claims an “S1solvent” or “S1” is an organic solvent with a water solubility of lessthan 15% characterized by a polarity related component of Hoy's cohesionparameter (delta-P) between 5 and 10 MPa^(1/2) and/or by a hydrogenbonding related component of Hoy's cohesion parameter (delta-H) between5 and 20 MPa^(1/2). In some exemplary embodiments of the invention, HCltends to selectively transfer to an S1 solvent upon contact therewith.

For purposes of this specification and the accompanying claims an “S2solvent” or “S2” is an organic solvent having a water solubility of atleast 30% and characterized by a delta-P greater than 8 MPa^(1/2) and/ora delta-H greater than 12 MPa^(1/2). In some exemplary embodiments ofthe invention, HCl tends to selectively transfer to an extractantincluding both S1 and S2 solvents upon contact therewith.

For purposes of this specification and the accompanying claims “extract”and “extraction” indicate bringing an extractant into contact with asubstrate and then separating an extract from an extracted substrate.

According to various exemplary embodiments of the invention anextraction may be on an indicate stream or fraction per se or on amodified stream or fraction. Optional modifications include, but are notlimited to, dilution, concentration, mixing with another stream orfraction, temperature adjustment, and filtration. Optionally, two ormore modifications may be performed prior to extraction.

“Woody materials” or “lignocellulosic materials” are an attractive andenvironment-friendly substrate for sugar production since they areobtained from renewable resources. Many non-food lignocellulosicmaterials are potential sources of soluble carbohydrates. Theselignocellulosic materials include, but are not limited to, wood andby-products of wood processing (e.g. chips, sawdust, and shavings) aswell as residual plant material from agricultural products and paperindustry byproducts (e.g. cellulose containing residues and/or paperpulp)

Residual plant material from agricultural products includes processingby-products and field remains.

Processing by-products include, but are not limited to, corn cobs, sugarcane bagasse, sugar beet pulp, empty fruit bunches from palm oilproduction, straw (e.g. wheat or rice), soy bean hulls, residual mealsfrom the vegetable oil industry (e.g. soybean, peanut, corn orrapeseed), wheat bran and fermentation residue from the beer and wineindustries.

Field remains include, but are not limited to, corn stover, post harvestcotton plants, post harvest soybean bushes and post harvest rapeseedplants.

Lignocellulosic materials also include “energy crops” such as switchgrass and broom grass which grow rapidly and generate low-cost biomassspecifically as a source of carbohydrates.

These lignocellulosic carbohydrate sources contain cellulose,hemicellulose and lignin as their main components and also containmineral salts (ashes) and lipophilic organic compounds, such as talloils. The degree and type of theses non-carbohydrate materials cancreate technical problems in production of soluble carbohydrates.

Lignocellulosic materials typically contain 65-80% cellulose andhemicelluloses on a dry matter basis. Cellulose and hemicellulose arepolysaccharides which can release carbohydrates suitable forfermentation and/or chemical conversion to products of interest if theyare hydrolyzed. Lignin is typically resistant to acid hydrolysis.

It will be appreciated that the various aspects described above relateto solution of technical problems associated with obtaining specificratios of disaccharides and/or higher oligomers relative to totalsaccharides produced by acid hydrolysis of a lignocellulosic substrate.

Alternatively or additionally, it will be appreciated that the variousaspects described above relate to solution of technical problemsassociated with the need for defined mixtures of sugars containingspecific disaccharides and/or higher oligomers. In many cases this“need” is defined by a specific downstream application.

In some exemplary embodiments of the invention, there is provided asugar mixture including: (i) monosaccharides; (ii) oligosaccharides in aratio to total saccharides ≧0.06; (iii) disaccharides in a ratio tototal saccharides ≧0.05; (iv) pentose in a ratio to total saccharides≧0.05; (v) at least one alpha-bonded di-glucose; and (vi) at least onebeta-bonded di-glucose.

Optionally, the mixture has a higher oligosaccharides in a ratio tototal saccharides ≦0.2.

Optionally, the mixture has a ratio of at least one of the alpha-bondeddi-glucose and the beta-bonded di-glucose relative to total saccharidesis ≧0.01.

Optionally, the mixture has a ratio of at least one of the alpha-bondeddi-glucose and the beta-bonded di-glucose relative to total saccharidesis ≧0.03.

Optionally, the alpha-bonded di-glucose includes at least one member ofthe group consisting of maltose, isomaltose and trehalose.

Optionally, the beta-bonded di-glucose includes at least one memberselected from the group consisting of gentiobiose, sophorose andcellobiose.

In some exemplary embodiments of the invention, there is provided amethod including (a) hydrolyzing a lignocellulosic material in a mediumcontaining HCl in a ratio to (HCl+water)≧0.37 to form a hydrolyzateincluding total saccharides in a ratio to (total saccharides+water)≧0.20by weight; (b) de-acidifying the hydrolyzate to form a de-acidifiedhydrolyzate including: (i) total saccharides in a ratio to (totalsaccharides+water)≧0.35 and; (ii) total disaccharides in a ratio tototal saccharides ≧0.05; and (c) adjusting a composition of thede-acidified hydrolyzate to form a mixture according to any of claims 1to 6.

In some exemplary embodiments of the invention, there is provided amethod including

(a) hydrolyzing a lignocellulosic material in a medium containing HCl ina ratio to (HCl+water)≧0.37 by weight to form a hydrolyzate includingtotal saccharides in a ratio to (total saccharides+water)≧0.20 byweight; (b) de-acidifying the hydrolyzate to form a de-acidifiedhydrolyzate including a mixture as described above.

Optionally, the hydrolyzing is conducted in a counter-current mode ofoperation.

Optionally, the hydrolyzing is conducted at a temperature of less than25° C.

Optionally, the lignocellulosic material includes softwood, for example,pine.

Optionally, the de-acidifying includes selective extraction of HCl andwater with an extractant including alcohol.

Optionally, the de-acidifying is conducted at a temperature of less than80° C.

In some exemplary embodiments of the invention, there is provided amethod including: (i) providing a preparation including HCl and a sugarmixture according to any of claims 1 to 6, and (ii) de-acidifying thepreparation to form a de-acidified preparation.

Optionally, the de-acidifying includes selective extraction of HCl withan extractant including alcohol.

Optionally, the de-acidifying is conducted at a temperature of less than80° C.

In some exemplary embodiments of the invention, there is provided amethod including: (a) providing a fermentor; and (b) fermenting a mediumincluding a sugar mixture as described above in the fermentor to producea fermentation product.

In some exemplary embodiments of the invention, there is provided amethod including: (a) providing a fermentor; and (b) fermenting a mediumincluding a de-acidified hydrolyzate according as described above or ade-acidified preparation as described above to produce a fermentationproduct.

Optionally, the fermentation product includes at least one memberselected from the group consisting of alcohols, carboxylic acids, aminoacids, monomers for the polymer industry and proteins.

Optionally, the method includes processing the fermentation product toproduce a consumer product selected from the group consisting ofdetergent, polyethylene-based products, polypropylene-based products,polyolefin-based products, polylactic acid (polylactide)-based products,polyhydroxyalkanoate-based products and polyacrylic-based products.

Optionally, the detergent includes a sugar-based surfactant, a fattyacid-based surfactant, a fatty alcohol-based surfactant, or acell-culture derived enzyme.

Optionally, the polyacrylic-based product is selected from plastics,floor polishes, carpets, paints, coatings, adhesives, dispersions,flocculants, elastomers, acrylic glass, absorbent articles, incontinencepads, sanitary napkins, feminine hygiene products, and diapers.

Optionally, the polyolefin-based products are selected from milk jugs,detergent bottles, margarine tubs, garbage containers, water pipes,absorbent articles, diapers, non wovens, HDPE toys and HDPE detergentpackagings.

Optionally, the polypropylene based products are selected from absorbentarticles, diapers and non wovens.

Optionally, the polylactic acid based products are selected frompackaging of agriculture products and of dairy products, plasticbottles, biodegradable products and disposables.

Optionally, the polyhydroxyalkanoate based products are selected frompackaging of agriculture products, plastic bottles, coated papers,molded or extruded articles, feminine hygiene products, tamponapplicators, absorbent articles, disposable nonwovens and wipes, medicalsurgical garments, adhesives, elastometers, films, coatings, aqueousdispersants, fibers, intermediates of pharmaceuticals and binders.

Optionally, the fermentation product includes at least one member of thegroup consisting of ethanol, butanol, isobutanol, a fatty acid, a fattyacid ester, a fatty alcohol and biodiesel.

Optionally, the method includes processing of the fermentation productto produce at least one product selected from the group consisting of anisobutene condensation product, jet fuel, gasoline, gasohol, dieselfuel, drop-in fuel, diesel fuel additive, and a precursor thereof.

Optionally, the gasahol is ethanol-enriched gasoline or butanol-enrichedgasoline.

Optionally, the product is selected from the group consisting of dieselfuel, gasoline, jet fuel and drop-in fuels.

In some exemplary embodiments of the invention, there is provided aconsumer product, a precursor of a consumer product, or an ingredient ofa consumer product produced from a fermentation product as describedabove.

In some exemplary embodiments of the invention, there is provided aconsumer product, a precursor of a consumer product, or an ingredient ofa consumer product including at least one fermentation product producedby a method as described above, wherein the fermentation product isselected from carboxylic and fatty acids, dicarboxylic acids,hydroxylcarboxylic acids, hydroxyl di-carboxylic acids, hydroxyl-fattyacids, methylglyoxal, mono-, di-, or poly-alcohols, alkanes, alkenes,aromatics, aldehydes, ketones, esters, biopolymers, proteins, peptides,amino acids, vitamins, antibiotics, and pharmaceuticals.

Optionally, the product is ethanol-enriched gasoline, jet fuel, orbiodiesel.

In some exemplary embodiments of the invention, there is providedconsumer product, a precursor of a consumer product, or an ingredient ofa consumer product according as described above, wherein the consumerproduct has a ratio of carbon-14 to carbon-12 of about 2.0×10⁻¹³ orgreater.

Optionally, the consumer product including an ingredient according asdescribed above and an additional ingredient produced from a rawmaterial other than lignocellulosic material.

Optionally, the ingredient and the additional ingredient produced from araw material other than lignocellulosic material are essentially of thesame chemical composition.

Optionally, the consumable product includes a marker molecule at aconcentration of at least 100 ppb.

Optionally, the marker molecule is selected from the group consisting offurfural, hydroxy-methyl furfural, products of furfural orhydroxy-mathylfurfural condensation, color compounds derived from sugarcaramelization, levulinic acid, acetic acid, methanol, galcturonic acid,and glycerol.

In some exemplary embodiments of the invention, there is provided amethod including:

(a) de-acidifying an acid hydrolyzate including total saccharides in aratio to (total saccharides+water)≧0.20 by weight to produce a sugarmixture with total saccharides in a ratio to (total saccharides+water)ratio≧0.35; the mixture including monosaccharides, the mixture havingdisaccharides in a ratio to total saccharides ≧0.05; and (b)enzymatically hydrolyzing the mixture with an enzyme capable ofcatalyzing hydrolysis of alpha bonds in the mixture so that at least 10%of the disaccharides are converted to monosaccharides; and (c)converting at least a portion of the saccharides to a conversionproduct.

Optionally, the sugar mixture includes higher oligosaccharides.

Optionally, at least 10% of the higher oligosaccharides are hydrolyzed.

Optionally, the acid hydrolyzate is the result of counter-currenthydrolysis.

Optionally, the acid hydrolyzate is the result of hydrolysis conductedat a temperature of less than 25° C.

Optionally, the de-acidifying includes extraction with an extractantincluding an alcohol.

Optionally, the de-acidifying is conducted at a temperature of less than80° C.

Optionally, the enzymatically hydrolyzing includes use of an enzymecapable of catalyzing hydrolysis of beta bonds.

Optionally, the enzyme includes at least one enzyme selected from thegroup consisting of amylases cellulases, hemicellulases,transglucosidases, glucoamylases, alpha-glucosidases and pullulanases.

Optionally, the enzymatically hydrolyzing includes use of an immobilizedenzyme.

Optionally, at least a portion of the converting is conductedsimultaneously with the enzymatically hydrolyzing.

Optionally, the total saccharides ratio to (total saccharides+water) is≧0.15 during the enzymatically hydrolyzing.

Optionally, the enzymatically hydrolyzing includes incubation of themixture with a microorganism.

Optionally, the converting includes fermentation.

Optionally, the sugar mixture includes at least one pentose in a ratioto total saccharides ≧0.05.

Optionally, the de-acidifying includes extracting the hydrolyzate, witha first extractant including an S1 solvent to form an HCl-carrying firstextract and an HCl-depleted sugar solution.

Optionally, the de-acidifying includes including chromatographicallyseparating the HCl-depleted sugar solution to produce a monosaccharideenriched monomer cut and an acid cut enriched in disaccharides andhigher oligosaccharides.

Optionally, the de-acidifying includes subsequently extracting theHCl-depleted sugar solution with a second extractant including S1 and asecond solvent (S2).

Optionally, the S1 of the extracting and the subsequently extractingeach independently include at least one member selected from the groupconsisting of alcohols, ketones and aldehydes having at least 5 carbonatoms and combinations thereof.

Optionally, the second extractant is characterized by at least one of:

a delta-P greater than the delta-P of the first extractant by at least0.2 MPa^(1/2); anda delta-H greater than the delta-H of the first extractant by at least0.2 MPa^(1/2).

Optionally, S2 includes at least one member selected from the groupconsisting of C₁-C₄ mono- or poly-alcohols, aldehydes and ketones.

Optionally, a ratio of HCl to total saccharides in the sugar mixture is≦0.03 by weight.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials are described below, methods and materials similar orequivalent to those described herein can be used in the practice of thepresent invention. In case of conflict, the patent specification,including definitions, will control. All materials, methods, andexamples are illustrative only and are not intended to be limiting.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying inclusion of the statedfeatures, integers, actions, ratios or components without precluding theaddition of one or more additional features, integers, actions, ratios,components or groups thereof. This term is broader than, and includesthe terms “consisting of” and “consisting essentially of” as defined bythe Manual of Patent Examination Procedure of the United States Patentand Trademark Office.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of architecture and/or computer science.

Percentages (%) and/or ratios of sugars (saccharides) to a totalmixture, as well as ratios of various sugars to one another (e.g.monosaccharides to disaccharides) are W/W (weight per weight) unlessotherwise indicated. Percentages (%) and/or ratios of HCl are alsoexpressed as W/W (weight per weight) unless otherwise indicate.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying figures.In the figures, identical and similar structures, elements or partsthereof that appear in more than one figure are generally labeled withthe same or similar references in the figures in which they appear.Dimensions of components and features shown in the figures are chosenprimarily for convenience and clarity of presentation and are notnecessarily to scale. The attached figures are:

FIG. 1 is a schematic overview of a system illustrating the industrialcontext of some exemplary embodiments of the invention;

FIGS. 2 a and 2 b are each simplified flow diagrams of methods accordingto exemplary embodiments of the invention;

FIG. 3 is a simplified flow diagram of a method according to anexemplary embodiment of the invention;

FIGS. 4 a and 4 b are each simplified flow diagrams of methods accordingto exemplary embodiments of the invention;

FIG. 5 is a simplified flow diagram of a method according to anexemplary embodiment of the invention; and

FIG. 6 is a simplified flow diagram of a portion of the method depictedin FIG. 5 in greater detail according to an exemplary embodiment of theinvention.

DETAILED DESCRIPTION OF EMBODIMENTS

Embodiments of the invention relate to sugar mixtures, their preparationand use. Specifically, some embodiments of the invention can be used assubstrates for microbial fermentation. Optionally, the specificmicroorganisms employed are selected to utilize one or more sugarspresent in the mixture. Alternatively or additionally, some embodimentsof the invention relate to adjusting one or more component ratios withina mixture to render the mixture more valuable for a specific downstreamapplication.

The principles and operation of methods according to exemplaryembodiments of the invention may be better understood with reference tothe drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Overview of Exemplary System

FIG. 1 is a simplified schematic diagram of a system for acid hydrolysisof a lignocellulosic substrate indicated generally as 100. Depictedsystem 100 includes a main hydrolysis reactor 110 adapted to receive alignocellulosic substrate input 112. Optionally, substrate 112 isprovided as wood chips, although any “woody material” as described inthe background can be used instead of wood.

Substrate 112 is brought into contact with a concentrated HCl solutionin reactor 110 and hemicellulose and/or cellulose in the substrate arehydrolyzed to produce a mixture of soluble sugars and residual lignin.These materials are collected separately as lignin stream 120 and sugarmixture 130, each of which contains a large amount of HCl.

Since the acid acts as a catalyst, it is not consumed in the process. Inaddition, residual acid content of the product and the co-productsshould be low in order to enable their use. Acid recovery from thehydrolyzate should be conducted under conditions minimizing thermaldegradation.

Details of exemplary hydrolysis methods and systems are described indetail in co-pending U.S. provisional applications 61/483,777 and61/487,319, each of which is fully incorporated herein by reference.

This application is primarily concerned with processing of sugar mixture130. The processing includes removal of HCl and/or adjustment of themixture to achieve one or more desired ratios of mixture components(e.g. disaccharides and/or monosaccharides). This processing isconducted in a sugar refining module, designated here generically as200.

Optionally, additional sugar mixture is recovered from lignin stream 120as described in co-pending U.S. provisional application U.S. 61/491,243which is fully incorporated herein by reference. In some exemplaryembodiments of the invention, this additional sugar mixture is routed torefining module 200. According to various exemplary embodiments of theinvention this additional sugar mixture increase a total sugar yieldand/or changes a composition of the mixture.

As will be explained in greater detail hereinbelow, refining module 200employs a flow of organic solvent 155 (solid arrows) to extract HCl 140(dashed arrows) from sugar mixture 130.

De-acidified sugars 230 are the primary product of refining module 200.Module 200 also produces a stream of HCl 140 mixed with solvent 155(depicted as parallel dashed and solid arrows respectively for clarity)which is routed to a solvent/HCl recovery module 150. Recovery module150 separates HCl 140 from solvent 155. In some exemplary embodiments ofthe invention, separation is by distillation. HCl 140 is recycled tohydrolysis reactor 110 and solvent 155 is recycled to refining module200.

De-acidified sugars 230 can be used in various industrial conversionprocesses as described hereinbelow. Optionally, additional adjustmentsare made on de-acidified sugar 230 prior to these conversion processesas described below.

Exemplary Sugar Mixtures:

Some exemplary embodiments of the invention relate to sugar mixtures. Insome exemplary embodiments of the invention, the mixtures can becharacterized as: containing monosaccharides (e.g. hexoses such asglucose and/or galactose and/or mannose) and having an oligosaccharidesto total saccharides ratio≧0.06. Optionally, the mixture includesdisaccharides and a disaccharides to total saccharides ratio≧0.05.Optionally, the mixture includes a pentose (e.g. xylose and/or arabinoseand/or ribose and/or lyxose) and a pentose to total saccharidesratio≧0.05. In some exemplary embodiments of the invention, the mixtureincludes at least one alpha-bonded di-glucose (glucose bonded toglucose) and/or at least one beta-bonded di-glucose. Optionally, themixture is characterized by a higher oligosaccharides to totalsaccharide ratio ≦0.2.

According to various exemplary embodiments of the invention thealpha-bonded di-glucose includes one or more of maltose, isomaltose andtrehalose and the beta-bonded di-glucose includes one or more ofgentiobiose, sophorose and cellobiose.

In some exemplary embodiments of the invention a ratio of at least oneof said alpha-bonded di-glucose and said beta-bonded di-glucose relativeto the total saccharide content is ≧0.01, optionally ≧0.03.

In some exemplary embodiments of the invention, the alpha-bondeddi-glucose includes maltose and/or isomaltose and/or trehalose and/orkojibiose and/or nigerose. In some exemplary embodiments of theinvention, the beta-bonded di-glucose includes gentiobiose and/orsophorose and/or cellobiose and/or laminaribiose and/or beta-trehalose.

In some exemplary embodiments of the invention, a proportion of thealpha-bonded di-glucose and/or the beta-bonded di-glucose is at least0.01, optionally at least 0.02, optionally at least 0.03 relative tototal saccharides.

According to some exemplary embodiments, the mixture includes multiplealpha-bonded di-glucoses and the combined proportion of thesedi-glucoses to total saccharides is at least 0.03. According to stillanother embodiment, the mixture includes multiple beta-bondeddi-glucoses and the combined proportion to total saccharides of thesebeta-bonded di-glucoses is at least 0.03.

Exemplary Methods to Make Such Mixtures:

FIG. 2 a is a simplified flow diagram depicting an exemplary method ofmaking sugar mixtures as described above indicated generally as 202.According to the depicted method a lignocellulosic material ishydrolyzed 210 in HCl. In some exemplary embodiments of the invention,hydrolysis 210 employs a medium containing a ratio of HCl to(HCl+water)≧0.37 by weight. Hydrolysis 210 yields a hydrolyzate 212comprising total saccharides to (total saccharides+water)≧0.20 byweight. The depicted method also includes de-acidifying 220 hydrolyzate212 to form a de-acidified hydrolyzate 222 comprising: (i) a ratio oftotal saccharides to (total saccharides+water)≧0.35 and; (ii) a ratio oftotal disaccharides to total saccharides ≧0.05. Optionally, an increasein saccharide concentration resulting from de-acidification 220contributes to an increase in value.

Method 202 also includes adjusting 230 a composition of de-acidifiedhydrolyzate 222 to form a sugar mixture as described above. According tovarious exemplary embodiments of the invention adjusting 230 includes,but is not limited to, one or more of concentration, dilution,polishing, active carbon treatment, ion exchange chromatography andenzymatic oligomerization (e.g. to form dimers). Optionally,glycosyltransferases are employed.

FIG. 2 b is a simplified flow diagram depicting an additional exemplarymethod of making sugar mixtures as described above indicated generallyas 204. Depicted method 204 includes hydrolyzing 210 to producehydrolyzate 212 as described for method 202.

However, in depicted method 204 de-acidifying 225 of hydrolyzate 212yields a de-acidified hydrolyzate comprising a mixture 240 according toan exemplary embodiment of the invention as described above.

With regards to methods 202 and/or 204 hydrolysis 210 is optionallyconducted in a counter-current mode of operation and/or at a temperatureof less than 25° C. In some exemplary embodiments of the invention, thelignocellulosic material hydrolyzed includes softwood, optionally pine.

According to various exemplary embodiments of methods 202 and/or 204dc-acidifying 220 or 225 includes selective extraction of HCl with analcohol. In some exemplary embodiments of the invention, the alcohol hasa water solubility of less than 15%, e.g. an alcohol with 5 to 8 carbonatoms. Optionally, some water is extracted with the HCl. Optionally; thede-acidifying is conducted at a temperature of less than 80° C.

Exemplary De-Acidification Methods

FIG. 3 is a simplified flow diagram depicting an exemplary method ofde-acidifying a sugar mixture as described above indicated generally as300. According to the depicted method a preparation including HCl and asugar mixture as described above is provided 310 and de-acidified 320.In some exemplary embodiments of the invention, de-acidifying 320includes selective extraction of HCl with an alcohol. Optionally,hexanol or 2-ethyl-1-hexanol is employed for this extraction.Optionally, de-acidifying 320 is conducted at a temperature of less than80° C., optionally less than 70° C. and optionally less than 60° C.

As used herein, “selective extraction” indicates that theHCl/carbohydrate ratio in the extract is greater than that ratio in theacidic hydrolyzate, optionally at least 5 fold greater, optionally atleast 10 fold.

In some exemplary embodiments of the invention, de-acidifying isconducted according to methods disclosed in co-pending Israeli patentapplication IL 206,152 which is fully incorporated herein by reference.

Exemplary Downstream Processing of Sugar Mixtures

FIG. 4 a is a simplified flow diagram depicting an exemplary method ofproducing a fermentation product from a sugar mixture as described aboveindicated generally as 402. According to the depicted method a fermentoris provided 410 and a media comprising a sugar mixture as describedabove is fermented 420 to produce a fermentation product 430.

FIG. 4 b is a simplified flow diagram depicting an exemplary method ofproducing a fermentation product from a sugar mixture as described aboveindicated generally as 404. According to the depicted method a fermentoris provided 410 and a media comprising a de-acidified hydrolyzate asdescribed above or a de-acidified preparation as described above isfermented 422 to produce a fermentation product 432.

According to various exemplary embodiments of the invention fermentationproduct 430 and/or 432 includes an alcohol and/or a carboxylic acidand/or an amino acid and/or a monomer for the polymer industry and/or aprotein.

One common fermentation product is ethanol, which may be useful, forexample, as a fuel. For example, ethanol may be added to gasoline toproduce “gasohol” as is commonly done in the United States, or used as afuel itself as commonly done in Brazil.

Optionally, the fermentation product is a protein. In some exemplaryembodiments of the invention, the protein is a heterologous protein anda disaccharide in the sugar mixture triggers an inducible regulatoryelement in a construct containing a sequence encoding the protein. Insome exemplary embodiments of the invention, increasing an amount and/orratio of a specific disaccharide in the sugar mixture contributes to anincreased yield of fermentation product 430 and/or 432 per unit of sugarmixture in the media fermented 420 and/or 422.

Additional Methods Including Enzymatic Hydrolysis

FIG. 5 is a simplified flow diagram depicting an exemplary method forthe production of a conversion product from a sugar mixture as describedabove indicated generally as 500. According to the depicted method, anacid hydrolyzate (as described above) including total saccharides to(total saccharides+water)≧0.20 by weight is de-acidified 510 to producea sugar mixture 520 with a total saccharide to (total saccharide+water)ratio≧0.35 including monosaccharides and having a disaccharides to totalsaccharides ratio≧0.05. This mixture is an exemplary sugar mixture asdescribed above.

Depicted exemplary method 500 also includes enzymatically hydrolyzing522 a sugar mixture with an enzyme capable of catalyzing hydrolysis ofalpha bonds in the mixture so that at least 10%, optionally at least20%, optionally at least 30% of the disaccharides are converted tomonosaccharides. The phrase “capable of catalyzing hydrolysis of alphabonds” indicates any enzyme with at least 5% of the activity of alphaamylase with respect to alpha bonds. Optionally, at least 10% of thedi-saccharides and higher saccharides are hydrolyzed to releaseadditional monosaccharides. Optionally, higher saccharides releaseadditional disaccharides.

In some exemplary embodiments of the invention, enzymaticallyhydrolyzing 522 is performed without having to concentrate dilutesaccharide solutions, i.e. the enzymatic hydrolysis is performed atrelatively high sugar concentration.

The depicted method also includes converting 530 at least a portion ofthe saccharides to a conversion product 540.

According to various exemplary embodiments of the invention convertingincludes fermentation and/or chemical conversion. Chemical conversioncan be, for example, catalytic conversion.

According to various exemplary embodiments of the invention, convertingand/or enzymatic hydrolysis may each be conducted in multiple stages invarious sequences. Optionally, one or more converting processes areconducted between enzymatic hydrolysis stages. Alternatively oradditionally, one or more enzymatic hydrolysis stages are conductedbetween converting processes.

In some exemplary embodiments of the invention, the sugar mixturecomprises oligosaccharides with at least three sugar units.

In some exemplary embodiments of the invention, the initial acidhydrolyzate results from counter-current hydrolysis. Optionally, theacid hydrolysis is conducted at a temperature of less than 25° C.

In some exemplary embodiments of the invention, de-acidifying 510includes extraction with an extractant including an alcohol. Optionally,de-acidifying 510 is conducted at a temperature of less than 80° C.

In some exemplary embodiments of the invention, enzymaticallyhydrolyzing 522 includes hydrolyzing beta bonds.

According to various exemplary embodiments of the inventionenzymatically hydrolyzing 522 includes use of at least one of one enzymebelonging to EC 3.2.1 according to the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)Enzyme Nomenclature Recommendations. This class of enzymes includes, butis not limited to, amylases cellulases, hemicellulases,transglucosidases, glucoamylases, alpha-glucosidases and pullulanases.Optionally, an alpha amylase and/or a beta amylase and/or a 1-4 alphaglucosidase are employed.

Optionally, one or more of these enzymes may be provided as part of anenzyme cocktail and/or a cellular extract including other undefinedenzymes.

In some exemplary embodiments of the invention, an immobilized enzyme isused for enzymatically hydrolyzing 522. Optionally, immobilizationcontributes to hydrolysis of a greater number of bonds per enzymemolecule.

In some exemplary embodiments of the invention, at least a portion ofconverting 530 is conducted simultaneously with enzymaticallyhydrolyzing 522. Optionally, this concurrent processing prevents buildupof hydrolysis products which might adversely effect enzyme hydrolysiskinetics. For purposes of this specification and the accompanying claimsthe term “simultaneously” is used in its art accepted sense (i.e.simultaneous saccharification and fermentation).

Optionally, a ratio of total saccharides to (totalsaccharides+water)≧0.15, optionally ≧0.2 is maintained during enzymatichydrolyzation.

According to some exemplary embodiments, enzymatically hydrolyzing 522includes a single reaction, or temporally and/or distinct reactions,which reactions differ, in the composition of enzymes employed and/or intemperature and/or in pH.

In some exemplary embodiments of the invention, enzymaticallyhydrolyzing 522 includes fermentation with micro-organisms that producethe desired enzyme(s).

Alternatively or additionally, converting 530 comprises fermentation.

Optionally, sugar mixture 520 includes at least one pentose and a ratiobetween the at least one pentose and total saccharides is ≧0.05 byweight.

Exemplary De-Acidification Processes

FIG. 6 is a simplified flow diagram which illustrates some exemplaryways in which de-acidification 510 of FIG. 5 can be accomplished.

In the depicted exemplary embodiments of the invention, de-acidifying510 includes extracting 511 the hydrolyzate with a first extractantincluding an S1 solvent to form an HCl-carrying first extract 612 and anHCl-depleted sugar solution 614. Optionally, the first extractantincludes 70, 80, 90, 95 or substantially 100% of the S1 solvent.However, HCl may be present in sugar solution 614 at an unacceptablelevel. If that is the case, one or more additional separation strategiescan optionally be employed.

According to various exemplary embodiments of the invention residual HClin HCl-depleted sugar solution 614 is removed by chromatographicseparation 620 and/or by a subsequent extraction 630 with a secondextractant including S1 and a second solvent (S2). Optionally, the totalsolvent concentration in the second extractant is 50 m 60, 70, 80, 90,95 or substantially 100% or intermediate percentages.

In those exemplary embodiments of the invention which employchromatographic separation 620 sugar solution 614 is separated into anHCl containing “acid cut” 624 which is enriched in disaccharides andhigher saccharides relative to total saccharides and a “monomer cut” 622enriched in monosaccharides relative to total saccharides. In someexemplary embodiments of the invention, acid cut 624 is subject tofurther treatment to separate saccharides from HCl and/or to adjust aration of monosaccharides to total saccharides. Exemplarychromatographic separation techniques are disclosed in co-pendingapplication IL 211093 which is fully incorporated herein by reference.

In those exemplary embodiments of the invention which employ asubsequent extraction 630, there is a selective transfer of HCl to thesecond extractant to form a second extract 634 and the de-acidifiedhydrolyzate 520 depicted here as de-acidified sugar mixture 632.Exemplary subsequent extraction techniques are disclosed in co-pendingPCT application IL2011/000130 which is fully incorporated herein byreference.

Optionally, the S1 solvent employed in the initial extracting 511 andthe subsequently extracting 630 includes a same solvent and/or adifferent solvent. S1 solvents employed in exemplary embodiments of theinvention include, but are not limited to alcohols (e.g. hexanol and2-ethyl hexanol), ketones and aldehydes having at least 5 carbon atomsand combinations thereof.

In some exemplary embodiments of the invention, the second extractant ischaracterized by a delta-P greater than the delta-P of the firstextractant by at least 0.2 MPa^(1/2) and/or a delta-H greater than thedelta-H of the first extractant by at least 0.2 MPa^(1/2).

According to various exemplary embodiments of the invention, S2 includesat least one member selected from the group consisting of C₁-C₄ mono- orpoly-alcohols, aldehydes and ketones.

In some exemplary embodiments of the invention, a ratio of HCl to totalsaccharides in the de-acidified hydrolyzate 520 and/or 632 is ≦0.03 byweight.

Exemplary Industrial Contexts of Downstream Processing

Potential downstream applications of soluble carbohydrates include, butare not limited to, production of bio-fuels (e.g. ethanol, butanol orhydrocarbons), use in the food industry (e.g. fermentation to citricacid or xanthan gum and conversion of xylose to xylitol for use as anartificial sweetener) and industrially useful monomers.

As new processes are developed for the production of alternative fuelssuch as fatty acid esters and hydrocarbons (directly formed byfermentation or produced by conversion of fermentation products), thedemand for soluble carbohydrates is expected to increase.

By way of example, sugar mixtures according to various exemplaryembodiments of the invention are expected to be useful in fermentorswhich employ inducible promoters such as that described in U.S. Pat. No.7,713,725 for example.

Alternatively or additionally, sugar mixtures according to variousexemplary embodiments of the invention are expected to be useful inproduction of fatty ester compositions such as that described in, forexample, US 2010/0071259.

Alternatively or additionally, sugar mixtures according to variousexemplary embodiments of the invention are expected to be useful inextractive fermentation such as described in, for example, WO2009/042950.

Alternatively or additionally, sugar mixtures according to variousexemplary embodiments of the invention are expected to be useful inproduction of fatty acid derivatives as described in, for example, WO2008/119082.

Alternatively or additionally, sugar mixtures according to variousexemplary embodiments of the invention are expected to be useful inproduction of peptides as described in, for example, U.S. Pat. No.7,595,173.

In summary, direct downstream products of sugar mixtures according tovarious exemplary embodiments of the invention resulting fromfermentation and/or conversion (e.g. alcohols, lactic acid, acrylic acidand antimicrobial peptide) are expected to give rise to a wide varietyof products resulting from further processing of these conversionproducts. Such products include, but are not limited to, automobile fuel(e.g. for automobiles and/or airplanes), diapers, plastic consumerproducts and paint.

Exemplary Consumer Products

Some exemplary embodiments of the invention, relate to consumer productsand their manufacture or preparation.

In some exemplary embodiments of the invention, a sugar mixtureaccording to one exemplary embodiment of the invention is converted to afermentation product according to one or more additional embodiments ofthe invention. Optionally, the fermentation product includes at leastone member selected from the group consisting of alcohols, carboxylicacids, amino acids, monomers for the polymer industry and proteins.

In some exemplary embodiments of the invention, the fermentation productis processed to produce a consumer product. Exemplary consumer productsinclude but are not limited to detergent, polyethylene-based products,polypropylene-based products, polyolefin-based products, polylactic acid(polylactide)-based products, polyhydroxyalkanoate-based products andpolyacrylic-based products.

In some exemplary embodiments of the invention, the detergent includes asugar-based surfactant and/or a fatty acid-based surfactant and/or afatty alcohol-based surfactant and/or a cell-culture derived enzyme.

In some exemplary embodiments of the invention, the polyacrylic-basedproduct includes a plastic and/or a floor polish and/or a carpet and/ora paint and/or a coating and/or an adhesive and/or a dispersion and/or aflocculants and/or an elastomer and/or acrylic glass and/or an absorbentarticles (e.g. incontinence pads, sanitary napkins, feminine hygieneproducts, and diapers).

Polyolefin-based products may be, for example, milk jugs, detergentbottles, margarine tubs, garbage containers, water pipes, absorbentarticles, diapers, non woven fabrics (e.g. pre-moistened towellettes),HDPE toys and HDPE detergent packagings.

In some exemplary embodiments of the invention, a polypropylene basedproduct is provided as an absorbent articles, optionally a diaper.

In other exemplary embodiments of the invention, a polypropylene basedproduct is provided as anon woven fabric item, optionally apre-moistened towellette.

Polylactic acid based products may be, for example, packaging ofagriculture or dairy products, plastic bottles, biodegradable productsand disposables.

Polyhydroxyalkanoate based products may be, for example, packaging ofagriculture products, plastic bottles, coated papers, molded or extrudedarticles, feminine hygiene products, tampon applicators, absorbentarticles, disposable nonwovens and wipes, medical surgical garments,adhesives, elastometers, films, coatings, aqueous dispersants, fibers,intermediates of pharmaceuticals and binders.

Optionally, the fermentation product includes at least one member of thegroup consisting of ethanol, butanol, isobutanol, a fatty acid, a fattyacid ester, a fatty alcohol and biodiesel. In some exemplary embodimentsof the invention, the fermentation product is processed to produce anisobutene condensation product and/or a jet fuel and/or gasoline and/orgasohol and/or diesel fuel and/or drop-in fuel and/or a diesel fueladditive and/or a precursor thereof. According to various exemplaryembodiments of the invention gasahol is ethanol-enriched gasoline orbutanol-enriched gasoline.

Optionally, the product is a diesel fuel. Optionally, the product isgasoline. Optionally, the product is jet fuel. Optionally, the productis a drop-in fuel.

According to various exemplary embodiments of the invention a consumerproduct, a precursor of a consumer product, or an ingredient of aconsumer product is produced from a fermentation product. Optionally,the consumer product, precursor of a consumer product, or ingredient ofa consumer product includes at least one fermentation product asdescribed hereinabove including one or more of carboxylic and fattyacids, dicarboxylic acids, hydroxylcarboxylic acids, hydroxyldi-carboxylic acids, hydroxyl-fatty acids, methylglyoxal, mono-, di-, orpoly-alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, esters,biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, andpharmaceuticals. In some exemplary embodiments of the invention, theproduct is ethanol-enriched gasoline, jet fuel, or biodiesel.

In some exemplary embodiments of the invention, a consumer product, oran ingredient of a consumer product according as described above has aratio of carbon-14 to carbon-12 of 1.8×10⁻¹³, optionally 2.0×10⁻¹³ orgreater.

In some exemplary embodiments of the invention, the consumer productincludes an ingredient as described above and an additional ingredientproduced from a raw material other than lignocellulosic material.Optionally, the ingredient and the additional ingredient are essentiallyof the same chemical composition.

Some exemplary embodiments of the invention relate to a consumableproduct as described above including a marker molecule at aconcentration of at least 100 ppb. The marker molecule optionallyincludes one or more of furfural, hydroxy-methyl furfural, products offurfural or hydroxy-mathylfurfural condensation, color compounds derivedfrom sugar caramelization, levulinic acid, acetic acid, methanol,galcturonic acid, and glycerol.

It is expected that during the life of this patent many EC 3.2.1 enzymeswill be characterized and the scope of the invention is intended toinclude all such new enzymes a priori.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

Specifically, a variety of numerical indicators have been utilized. Itshould be understood that these numerical indicators could vary evenfurther based upon a variety of engineering principles, materials,intended use and designs incorporated into the invention. Additionally,components and/or actions ascribed to exemplary embodiments of theinvention and depicted as a single unit may be divided into subunits.Conversely, components and/or actions ascribed to exemplary embodimentsof the invention and depicted as sub-units/individual actions may becombined into a single unit/action with the described/depicted function.

Alternatively, or additionally, features used to describe a method canbe used to characterize an apparatus and features used to describe anapparatus can be used to characterize a method.

It should be further understood that the individual features describedhereinabove can be combined in all possible combinations andsub-combinations to produce additional embodiments of the invention. Theexamples given above are exemplary in nature and are not intended tolimit the scope of the invention which is defined solely by thefollowing claims. Specifically, the invention has been described in thecontext of ethanol production but is widely applicable to anyfermentation or conversion process.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

The terms “include”, and “have” and their conjugates as used herein mean“including but not necessarily limited to”.

Additional objects, advantages, and novel features of variousembodiments of the invention will become apparent to one ordinarilyskilled in the art upon examination of the following examples, which arenot intended to be limiting. Additionally, each of the variousembodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below findsexperimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Materials and Methods

The following materials and methods are used in performance ofexperiments described in examples hereinbelow:

Enzymes:

Some enzymes (Examples 1-3) were from Novozymes (Novozymes A/S;Denmark):

-   -   Spyrizyme Fuel HS: Glucoamylase with a 1-4 and 1-6 activity;        1425 AGU/g declared activity; Light to dark-brown liquid.    -   Liquozyme SC DS: α-Amylase with 1-4 activity; 240 KNU-S/g        declared activity; Amber liquid.    -   Manufacturer's instructions are:    -   Spyrizyme Fuel HS: 0.025-0.035% (w/w) grain “as is”    -   Liquozyme SC DS: n.a.    -   Cellic CTec 1.5%, 3.0%, and 15% w/w (g enzyme/g cellulose).    -   Cellic HTech 0.1-0.5% (w/w TS).

Additional enzymes were obtained from Genencor (Danisco; Genencor,Beloit Wis., USA):

Spezyme Alpha: <1% α-Amylase activity 13,775 (Alpha Amylase Units)/g(min.), thermostable, dark-brown liquid, optimal pH 5.5-6.5.

Transglucosidase L-2000: 1-5% Active enzyme, having α 1-4 activity,yellow-brown liquid, optimal pH 4.5-5.5.

The recommended usages of enzymes (A. Kelley, Genencor) are:

Spezyme Alpha: 0.025%

Transglucosidase L-2000: 0.01%

Accelerase BG: 8%

Accelerase DUET: 8%

Hydrolyzates:

Hydrolyzates from Pinewood were employed as a substrate for enzymaticanalyses. The hydrolyzates were adjusted to contain a desired % TS(Total Suaccharides). All experiments were conducted at pH 4.0-5.1 atvarious temperatures from 50 to 85° C. Loading doses of the enzymes werealso varied.

In all of the enzymatic hydrolysis experiments time 0 (hrs.) refers tothe blank (without enzyme).

Hydrolyzates from pinewood, sugarcane bagasse and eucalyptus wood werealso subject to detailed analyses of monosaccharide and disaccharideconcentrations.

Control of Reaction Conditions:

The hydrolyzate was diluted with deionized (DI) water and buffer to adesired TS % and pH. The enzyme solution was added and the flask waskept at a selected temperature with stirring throughout incubation.Samples were withdrawn according to an experimental schedule and theenzyme was denaturated by heating and filtered out.

Saccharide Analyses:

Saccharide composition of the samples was analyzed by HPLC using aVarian Prostar® and a Rezex RSO-Oligosaccharide Ag+, 10×200 mm columnand pre-column in the following conditions:

Column Temp.: 80° C., Mobile Phase: Water (HPLC grade), Flow Rate: 0.3mL/min, Injection: 5-10 μL (depending on sugars conc.), Detector: RI,Detector Temp.: 40° C. The DP groups HPLC results are given in area %,the x-axis in the graphs represents time (hrs) and the y-axis area %.

DP stands for degree of polymerization, so that DP1 refers to allmono-saccharides, DP2 are all di-saccharides, DP3 are alltri-saccharides and DP>3 refers to oligosaccharides containing more thanthree sugar units (DP groups given as % area).

Example 1 Exemplary Enzymatic Hydrolysis of a De-Acidified Hydrolyzatewith 30% TS Using Spirizyme

In order to examine the influence of enzyme amounts on totalmonosaccharide yield, enzyme amount was titrated and efficiency ofenzymatic hydrolysis was measured.

In a first series of enzymatic hydrolysis experiment Spyrizyme Fuel HSand Liquozyme SC DS were employed.

A pinewood hydrolyzate with 30% TS at pH 4.9 was hydrolyzed with 3 mgenzyme solution/1 gr 100% sugar at a temperature of 62° C. with stirring(enzyme was 0.3%). Results are summarized in Table 1.

TABLE 1 Spirizyme Fuel HS (3 mg enzyme solution/1 gr 100% sugar) DP > 3Time (Hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 45.7 31.813.7 8.8 2.5 51.2 31.5 12.2 5.0 4.0 51.4 31.5 11.8 5.3 24 56.2 28.2 11.04.6

In order to assess the impact of increasing the amount of enzyme, 33 mgenzyme solution/1 gr 100% sugar were employed. Results are summarized inTable 2.

TABLE 2 Spirizyme Fuel HS (33 mg enzyme solution/1 gr 100% sugar) DP > 3Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 45.7 31.813.7 8.8 2.5 64.0 26.8 7.0 2.3 4.0 65.1 26.2 6.4 2.3

Results summarized in Tables 1 and 2 demonstrate a trend towardsincreased proportion of DP1 and DP2 saccharides at the expense of DP3and DP>3 saccharides. This trend increased as a function of time.

In order to assess the impact of increasing the amount of enzyme evenfurther, 65.6 mg enzyme solution/1 gr 100% sugar were employed. Resultsare summarized in Table 3.

TABLE 3 Spirizyme Fuel HS (65.6 mg enzyme solution/1 gr 100% sugar) DP >3 Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 45.7 31.813.7 8.8 2.5 68.2 26.8 7.0 2.3 4.0 68.1 26.2 6.4 2.3

Results presented in Table 3 indicate that it is possible to increasethe yield of monosaccharides even further.

In order to assess the impact of increasing the amount of enzyme evenfurther, 282.6 mg enzyme solution/1 gr 100% sugar were employed. Resultsare summarized in Table 4.

TABLE 4 Spirizyme Fuel HS (282.6 mg enzyme solution/1 gr 100% sugar)DP > 3 Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 45.731.8 13.7 8.8 2.5 70.6 22.4 5.6 1.5 4.0 71.1 21.6 5.5 1.8

Results summarized in Tables 1 to 4 indicate that enzymes capable ofhydrolyzing alpha bonds can be employed to increase monosaccharideconcentrations in acid hydrolyzates of cellulose. The enzyme dose of282.6 mg gave the highest yield of mono-saccharides.

However, other conditions may contribute to enzymatic hydrolysisresults. Exemplary other conditions include, but are not limited to,reaction temperature, exact nature of the sugars in the hydrolyzate interms of both bond type and oligomer length distribution.

Example 2 Influence of % TS on Enzymatic Hydrolysis of a De-AcidifiedHydrolyzate Using Spirizyme

In order to examine the influence of % TS in the substrate on totalmonosaccharide yield, enzyme amount was titrated again against ahydrolyzate similar to that used in Example 1, but with decreasingamounts of % TS and efficiency of enzymatic hydrolysis was measured.

A pinewood hydrolyzate with 15% TS at pH 5.1 was hydrolyzed with 72.8 mgenzyme solution/1 gr 100% sugar at a temperature of 60° C. with stirring(enzyme ˜243 of manufacturer's recommendation). Results are summarizedin Table 5. This experiment confirms results presented in Table 2 ofExample 1 and suggests that more dilute sugar solutions are moreamenable to enzymatic hydrolysis.

TABLE 5 Spirizyme Fuel HS (72.8 mg enzyme solution/1 gr 100% sugar) DP >3 Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 45.7 31.813.7 8.8 2.5 71.3 20.6 6.1 2.1 4.0 71.8 21.0 4.9 2.3 5.8 71.8 20.2 5.72.3 24 71.1 24.3 3.4 1.2 50 76.0 17.9 4.7 1.4

A pinewood hydrolyzate with 5% TS at pH 5.0 was hydrolyzed with 67 mgenzyme solution/1 gr 100% sugar at a temperature of 62° C. with stirring(enzyme ˜233 times manufacturer's recommendation). Results aresummarized in Table 6. This experiment confirms results presented inTable 2 of Example 1 and suggests that more dilute sugar solutions aremore amenable to enzymatic hydrolysis.

TABLE 6 Spirizyme Fuel HS (67 mg enzyme solution/1 gr 100% sugar) DP > 3Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 49.8 31.312.9 6.1 2.5 72.0 20.4 5.4 2.2 24 75.4 17.8 5.0 1.8 50 74.7 19.7 4.3 1.372 77.8 16.2 4.8 1.2 96 77.1 15.7 4.9 2.3

Results presented in table 6 confirm that enzymes capable of hydrolyzingalpha bonds can be employed to increase monosaccharide concentrations inacid hydrolyzates of cellulose.

Comparison of results presented in table 6 with those presented inTables 2 and 5 suggests that dilution of sugar solutions prior toenzymatic hydrolysis contributes to an increase in monosaccharide yield.

Again, the exact nature of the sugars in the hydrolyzate in terms ofboth bond type and oligomer length distribution may influence the totalyield of monosaccharides.

Example 3 Enzymatic Hydrolysis of a De-acidified Hydrolyzate with 30% TSUsing Liquozyme SC DS

In order to determine whether the results presented above were enzymespecific an additional experiment was conducted on a 30% TS hydrolyzateusing Liquozyme. A pinewood hydrolyzate with 30% TS at pH 6.0 washydrolyzed with 282.6 mg enzyme solution/1 gr 100% sugar at atemperature of 85° C. with stirring (enzyme ˜942 times manufacturer'srecommendation for Spirizyme). Results are summarized in Table 7. Thisexperiment confirms results presented in Table 2 of Example 1 andsuggests that more dilute sugar solutions are more amenable to enzymatichydrolysis.

TABLE 7 Liquozyme SC DS (282.6 mg enzyme solution/1 gr 100% sugar) DP >3 Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 45.7 31.813.7 8.8 4.0 59.8 27.1 8.4 4.5

Results presented in table 7 indicate that the ability of enzymescapable of hydrolyzing alpha bonds to increase monosaccharideconcentrations in acid hydrolyzates of cellulose is not specific toSpirizyme Fuel HS.

These results also suggest that Liquozyme SC DS provides a higher yieldof mono-saccharides than Spirizyme Fuel HS at 282.6 mg enzyme solution/1gr 100% sugar (see Table 4 for comparison). However, the desirability ofone enzyme over another may also be influence by other considerationsincluding, but not limited to, availability, purity, bond specificityand price.

Example 4 Enzymatic Hydrolysis of a De-Acidified Sugarcane BagasseHydrolyzate with Various Enzymes

In order to examine the possibility of applying enzymatic hydrolysis tohydrolyzates from non-wood substrates, a series of experiments wasconducted on hydrolyzates prepared from sugar cane bagasse. Thehydrolyzates used contained different TS (5%, 20%), the experiments wereconducted at pH 4.5-5.1, and at various temperatures (50° C. and 60°C.). The loading doses of the enzymes were also varied.

A first experiment was conducted using Spezyme Alpha at a concentrationof 34.2 mg enzyme solution/1 gr 100% sugar, at a temperature of 60° C.and at pH 5.1, applied to a bagasse hydrolyzate with 20% TS. Results aresummarized in Table 8.

TABLE 8 Spezyme Alpha applied to Baggase hydrolyzate TS 20% (34.2 mgenzyme solution; about 137 times manufacturer's recommendation) DP > 3Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 52.0 35.410.6 1.9 2.5 52.0 34.9 10.8 2.3 4.0 53.6 34.2 10.1 2.1 24 52.4 35.1 10.32.2 50 51.7 35.1 10.9 2.3

An additional experiment was conducted using Transglucosidase L-2000 ata concentration of 14.8 mg enzyme/1 gr 100% sugar at a temperature of60° C. at pH 5.0 applied to a bagasse hydrolyzate with 20% TS. Resultsare summarized in Table 9.

TABLE 9 Transglucosidase L-2000 applied to Baggase hydrolyzate TS 20%(14.8 mg enzyme; about 148 times manufacturer's recommendation) DP > 3Time (hr) DP1 (% area) DP2 (% area) DP3 (% area) (% area) 0 52.0 35.410.6 1.9 2.5 57.9 29.7 10.0 2.4 4.0 62.0 26.8 9.1 2.2 24 62.7 27.0 8.51.8 50 62.2 27.1 8.7 2.0

Results summarized in Tables 8 and 9 indicate that TransglucosidaseL-2000 provides an increase in mono-saccharides in the bagassehydrolyzate while Spezyme Alpha does not provide such an increase.

The results from Transglucosidase L-2000 (Table 9) confirm that enzymescapable of hydrolyzing alpha bonds can be used to increase the yield ofmonosaccharides.

The negative results from Spezyme Alpha presented in table 8 suggestthat there may be inhibitors present in the hydrolyzate. At this stageit is not clear whether these inhibitors are specific to the substratesubject to the initial acid hydrolysis (e.g. sugar cane bagasse asopposed to pine wood) or are specific to the enzyme.

Example 5 Saccharide Composition of Hydrolyzates of a First Pine Wood

In order to obtain a sugar mixture, dry first pinewood was introducedinto a six stage hydrolysis reactor series in a counter-currentoperation as described in co-pending U.S. provisional application61/48377 filed May 9, 2011 and entitled “Hydrolysis systems andmethods”. This application is fully incorporated herein by reference.

Briefly, an aqueous solution of 42% HCl was introduced continually at atemperature of 10-15° C. for 24 hours. The hydrolyzate was collected,HCl was removed by extraction and the deacidified hydrolyzate wasconcentrated to give a sugar composition. The composition was analyzedby HPLC, the sample's total sugars was 74.3%. Analysis results ofmonosaccharides and disaccharides are summarized in Tables 10 and 11respectively. The results are calculated as % from sample's refractivetotal saccharides (%/RTS).

TABLE 10 Monosaccharides in hydrolyzate of the first pine wood RhamnoseArabinose Galactose Glucose Xylose Mannose Sum 0.1 1.6 2.7 27.7 7.0 7.446.5

TABLE 11 Disaccharides in hydrolyzate of the first pine wood TrehaloseIsomaltose Gentiobiose Cellobiose Nigerose Sophorose Other DP2 Sum 1.44.8 1.0 0.7 0.4 0.2 14.5 22.9

Results summarized in table 10 illustrate the presence of pentoses suchas Arabinose and Xylose and Rhamnose (de-oxy pentose). Since thesepentoses are prone to degradation under harsh acidic conditions, theirpresence suggests that the counter current design of the hydrolysisreactor contributes to a “pentose sparing” effect.

Results summarized in Table 11 illustrate the presence of alpha-bondeddi-glucose such as Trehalose and Isomaltose, as well as the presence ofbeta-bonded di-glucose such as Gentiobiose and Sophorose.

Example 6 Saccharide Composition of Hydrolyzates of a Second Pine Wood

Similarly, a second pine wood was hydrolyzed, deacidified andconcentrated to 77% TS.

Analysis results of monosaccharides and disaccharides are presented inTables 12 and 13 respectively. The results are calculated as % fromsample's refractive total saccharides (%/RTS).

TABLE 12 monosaccharides in hydrolyzate of the second wood ArabinoseGalactose Glucose Xylose Mannose Sum 0.3 0.8 36 8 1.0 46

TABLE 13 results of disaccharides in hydrolyzate of the second woodTreha- Isomal- Genti- Cello- Other lose tose obiose biose NigeroseMaltose DP2 Sum 1.1 6.6 2.1 2.1 0.4 0.4 28 41

The second pine wood also contained 16.7% higher oligosaccharides.

Results summarized in tables 12 and 13 confirm the presence of pentose,of alpha-bonded di-glucose and of beta-bonded di-glucose. There aredifferences in the saccharide profiles of hydrolyzates of the two pinesources.

Example 7 Saccharide Composition of Hydrolyzate Prepared from Non-PineWood Substrates

In order to examine the effect of substrate composition on hydrolyzatecomposition in terms of specific sugars, deacidified hydrolyzatesprepared from sugar cane bagasse and Eucalyptus wood were analyzed as inExample 6.

Analysis results of monosaccharides and disaccharides from the sugarcane bagasse hydrolyzate are presented in Tables 14 and 15 respectively.The results are calculated as % from sample's refractive totalsaccharides (%/RTS).

TABLE 14 results of monosaccharides in hydrolyzate of sugar cane bagasseArabinose Galactose Glucose Xylose Mannose Fructose Sum 2.2 7.2 48.7 4.94.8 2.4 70.2

TABLE 15 results of disaccharides in hydrolyzate of sugar cane bagasseIsomaltose Gentiobiose Maltose Other DP2 Sum 4.5 0.6 2.9 27 35

Analysis results of monosaccharides and disaccharides from theeucalyptus wood hydrolyzate are presented in Tables 16 and 17respectively. The results are calculated as % from sample's refractivetotal saccharides (%/RTS).

TABLE 16 results of monosaccharides in hydrolyzate of Eucalyptus woodArabinose Galactose Glucose Xylose Mannose Fructose Sum 2.6 7.24 46.18.27 5.83 3.38 73.42

TABLE 17 results of disaccharides in hydrolyzate of sugar Eucalyptuswood Isomaltose Cellobiose Maltose Other DP2 Sum 7.1 1.85 2.16 11.11 22

Results presented in tables 14 to 17, taken together with resultspresented in tables 10 to 13 suggest that the substrate used for theinitial acid hydrolysis can influence the saccharide profile of theresultant de-acidified hydrolyzate.

1. A sugar mixture comprising: (i) monosaccharides; (ii)oligosaccharides in a ratio to total saccharides ≧0.06; (iii)disaccharides in a ratio to total saccharides ≧0.05; (iv) pentose in aratio to total saccharides ≧0.05; (v) at least one alpha-bondeddi-glucose; and (vi) at least one beta-bonded di-glucose.
 2. A mixtureaccording to claim 1, having higher oligosaccharides in a ratio to totalsaccharides ≦0.2.
 3. A mixture according to claim 1, wherein a ratio ofat least one of said alpha-bonded di-glucose and said beta-bondeddi-glucose relative to total saccharides is ≧0.01.
 4. A mixtureaccording to claim 3, wherein a ratio of at least one of saidalpha-bonded di-glucose and said beta-bonded di-glucose relative tototal saccharides is ≧0.03.
 5. A mixture according to claim 1, whereinsaid alpha-bonded di-glucose includes at least one member of the groupconsisting of maltose, isomaltose and trehalose.
 6. A mixture accordingto claim 1, wherein said beta-bonded di-glucose includes at least onemember selected from the group consisting of gentiobiose, sophorose andcellobiose. 7-14. (canceled)
 15. A method comprising: (i) providing apreparation comprising HCl and a sugar mixture according to claim 1, and(ii) de-acidifying said preparation to form a de-acidified preparation.16. A method according to claim 15, wherein said de-acidifying comprisesselective extraction of HCl with an extractant including an alcohol. 17.A method according to claim 15, wherein said de-acidifying is conductedat a temperature of less than 80° C.
 18. A method comprising: (a)providing a fermentor; and (b) fermenting a medium comprising a sugarmixture according to claim 1 in said fermentor to produce a fermentationproduct.
 19. (canceled)
 20. A method according to claim 18, wherein saidfermentation product includes at least one member selected from thegroup consisting of alcohols, carboxylic acids, amino acids, monomersfor the polymer industry and proteins.
 21. A method according to claim18, comprising processing said fermentation product to produce aconsumer. 22-27. (canceled)
 28. A method according to claim 18, whereinsaid fermentation product includes at least one member of the groupconsisting of ethanol, butanol, isobutanol, a fatty acid, a fatty acidester, a fatty alcohol and biodiesel.
 29. A method according to claim28, comprising processing of said fermentation product. 30-31.(canceled)
 32. A consumer product, a precursor of a consumer product, oran ingredient of a consumer product produced from a fermentation productaccording to claim
 18. 33. (canceled)
 34. A consumer product accordingto claim
 32. 35. The consumer product, a precursor of a consumerproduct, or an ingredient of a consumer product according to claim 32,wherein said consumer product has a ratio of carbon-14 to carbon-12 ofabout 2.0×10⁻¹³ or greater.
 36. A consumer product comprising aningredient according to claim 32 and an additional ingredient producedfrom a raw material other than lignocellulosic material.
 37. (canceled)38. A consumer product according to claim 32, comprising a markermolecule at a concentration of at least 100 ppb.
 39. (canceled)
 40. Amethod comprising: (a) de-acidifying an acid hydrolyzate comprisingtotal saccharides in a ratio to (total saccharides+water)≧0.20 by weightto produce a sugar mixture with total saccharides in a ratio to (totalsaccharides+water) ratio≧0.35; said mixture comprising monosaccharides,said mixture having disaccharides in a ratio to total saccharides ≧0.05;and (b) enzymatically hydrolyzing said mixture with an enzyme capable ofcatalyzing hydrolysis of alpha bonds in said mixture so that at least10% of said disaccharides are converted to monosaccharides; and (c)converting at least a portion of said saccharides to a conversionproduct. 41-61. (canceled)